This invention relates generally to viral vector, and more particularly, to recombinant viral vectors useful for gene delivery.
Adeno-associated viruses are small, single-stranded DNA viruses which require helper virus to facilitate efficient replication [K. I. Berns, Parvoviridae: the viruses and their replication, p. 1007-1041, in F. N. Fields et al., Fundamental virology, 3rd ed., vol. 2, (Lippencott-Raven Publishers, Philadelphia, Pa.) (1995)]. The 4.7 kb genome of AAV is characterized by two inverted terminal repeats (ITR) and two open reading frames which encode the Rep proteins and Cap proteins, respectively. The Rep reading frame encodes four proteins of molecular weight 78 kD, 68 kD, 52 kD and 40 kD. These proteins function mainly in regulating AAV replication and integration of the AAV into a host cell""s chromosomes. The Cap reading frame encodes three structural proteins in molecular weight 85 kD (VP 1), 72 kD (VP2) and 61 kD (VP3) [Berns, cited above]. More than 80% of total proteins in AAV virion comprise VP3. The two ITRs are the only cis elements essential for AAV replication, packaging and integration. There are two conformations of AAV ITRs called xe2x80x9cflipxe2x80x9d and xe2x80x9cflopxe2x80x9d. These differences in conformation originated from the replication model of adeno-associated virus which use the ITR to initiate and reinitiate the replication [R. O. Snyder et al., J. Virol., 67:6096-6104 (1993); K. I. Berns, Microbiological Reviews, 54:316-329 (1990)].
AAVs have been found in many animal species, including primates, canine, fowl and human [F. A. Murphy et al., xe2x80x9cThe Classification and Nomenclature of Viruses: Sixth Report of the International Committee on Taxonomy of Virusesxe2x80x9d, Archives of Virology, (Springer-Verlag, Vienna) (1995)]. In addition to five known primate AAVs (AAV-1 to AAV-5), AAV-6, another serotype closely related to AAV-2 and AAV-1 has also been isolated [E. A. Rutledge et al., J. Virol., 72:309-319 (1998)]. Among all known AAV serotypes, AAV-2 is perhaps the most well-characterized serotype, because its infectious clone was the first made [R. J. Samulski et al., Proc. Natl. Acad. Sci. USA, 79:2077-2081 (1982)]. Subsequently, the full sequences for AAV-3A, AAV-3B, AAV-4 and AAV-6 have also been determined [Rutledge, cited above; J. A. Chiorini et al., J. Virol., 71:6823-6833 (1997); S. Muramatsu et al., Virol., 221:208-217 (1996)]. Generally, all AAVs share more than 80% homology in nucleotide sequence.
A number of unique properties make AAV a promising vector for human gene therapy [Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992)]. Unlike other viral vectors, AAVs have not been shown to be associated with any known human disease and are generally not considered pathogenic. Wild type AAV is capable of integrating into host chromosomes in a site specific manner [R. M. Kotin et al., Proc. Natl. Acad. Sci. USA, 87:2211-2215 (1990) R. J. Samulski, EMBO J., 10(12):3941-3950 (1991)]. Recombinant AAV vectors can integrate into tissue cultured cells in chromosome 19 if the rep proteins are supplied in trans [C. Balague et al., J. Virol., 71:3299-3306 (1997); R. T. Surosky et al., J. Virol., 71:7951-7959 (1997)]. The integrated genomes of AAV have been shown to allow long term gene expression in a number of tissues, including, muscle, liver, and brain [K. J. Fisher, Nature Med., 3(3):306-312 (1997); R. O. Snyder et al., Nature Genetics, 16:270-276 (1997); X. Xiao et al., Experimental Neurology, 144:113-124 (1997); Xiao, J. Virol., 70(11):8098-8108 (1996)].
AAV-2 has been shown to be present in about 80-90% of the human population. Earlier studies showed that neutralizing antibodies for AAV-2 are prevalent [W. P. Parks et al., J. Virol., 2:716-722 (1970)]. The presence of such antibodies may significantly decrease the usefulness of AAV vectors based on AAV-2 despite its other merits. What are needed in the art are vectors characterized by the advantages of AAV-2, including those described above, without the disadvantages, including the presence of neutralizing antibodies.
In one aspect, the invention provides an isolated AAV-1 nucleic acid molecule which is selected from among SEQ ID NO: 1, the strand complementary to SEQ ID NO: 1, and cDNA and RNA sequences complementary to SEQ ID NO: 1 and its complementary strand.
In another aspect, the present invention provides AAV ITR sequences, which include the 5xe2x80x2 ITR sequences, nt 1 to 143 of SEQ ID NO: 1; the 3xe2x80x2 ITR sequences, nt 4576 to 4718 of SEQ ID NO: 1, and fragments thereof.
In yet another aspect, the present invention provides a recombinant vector comprising an AAV-1 ITR and a selected transgene. Preferably, the vector comprises both the 5xe2x80x2 and 3xe2x80x2 AAV-1 ITRs between which the selected transgene is located.
In still another aspect, the invention provides a recombinant vector comprising an AAV-1 P5 promoter having the sequence of nt 236 to 299 of SEQ ID NO: 1 or a functional fragment thereof.
In a further aspect, the present invention provides a nucleic acid molecule encoding an AAV-1 rep coding region and an AAV-1 cap coding region. In still another aspect, the present invention provides a host cell transduced with a recombinant viral vector of the invention. The invention further provides a host cell stably transduced with an AAV-1 P5 promoter of the invention.
In still a further aspect, the present invention provides a pharmaceutical composition comprising a carrier and a vector of the invention.
In yet another aspect, the present invention provides a method for AAVxe2x80x94mediated delivery of a transgene to a host involving the step of delivering to a selected host a recombinant viral vector comprising a selected transgene under the control of sequences which direct expression thereof and an adeno-associated virus 1 (AAV-1) virion.
In another aspect, the invention provides a method for in vitro production of a selected gene product using a vector of the invention.
Other aspects and advantages of the invention will be readily apparent to one of skill in the art from the detailed description of the invention.